Preclinical evaluation of the CDK4/6 inhibitor palbociclib in combination with a PI3K or MEK inhibitor in colorectal cancer

ABSTRACT Background Studies have demonstrated the efficacy of Palbociclib (CDK 4/6 inhibitor), Gedatolisib (PI3K/mTOR dual inhibitor) and PD0325901 (MEK1/2 inhibitor) in colorectal cancer (CRC), however single agent therapeutics are often limited by the development of resistance. Methods We compared the anti-proliferative effects of the combination of Gedatolisib and Palbociclib and Gedatolisib and PD0325901 in five CRC cell lines with varying mutational background and tested their combinations on total and phosphoprotein levels of signaling pathway proteins. Results The combination of Palbociclib and Gedatolisib was superior to the combination of Palbociclib and PD0325901. The combination of Palbociclib and Gedatolisib had synergistic anti-proliferative effects in all cell lines tested [CI range: 0.11–0.69] and resulted in the suppression of S6rp (S240/244), without AKT reactivation. The combination of Palbociclib and Gedatolisib increased BAX and Bcl−2 levels in PIK3CA mutated cell lines. The combination of Palbociclib and Gedatolisib caused MAPK/ERK reactivation, as seen by an increase in expression of total EGFR, regardless of the mutational status of the cells. Conclusion This study shows that the combination of Palbociclib and Gedatolisib has synergistic anti-proliferative effects in both wild-type and mutated CRC cell lines. Separately, the phosphorylation of S6rp may be a promising biomarker of responsiveness to this combination.


Introduction
Colorectal cancer (CRC) is the second most common cause of cancer death in Europe. It is estimated that by 2030 the global burden of CRC will increase by 60%, with 2.2 million new cases and one million deaths worldwide. 1 In the metastatic CRC setting, compound agent chemotherapies given in combination with anti-EGFR (epidermal growth factor receptor) or anti-VEGF (vascular endothelial growth factor) monoclonal antibodies are standard clinical practice, providing improvement in patient outcomes reaching median overall survival (OS) between 29 and 36 months (in patients with RAS wildtype disease). 2,3 However, more than 50% of patients eventually relapse and subsequent treatment options rarely offer high clinical impact, especially in patients with RAS mutations. 4 Treatment for CRC is complex and often limited by resistance to therapy, which can be intrinsic or acquired. The crosstalk between the PI3K/AKT/mTOR (phosphatidylinositol −3-kinase/acutely transforming retrovirus/mammalian target of rapamycin) and MAPK/ERK (mitogen activated protein kinase) pathways is recognized as a key mechanism of resistance to oncology therapy. 5 Other mechanisms leading to the development of resistance include 1) Formation of new secondary site resistance mutations within the target kinase; 2) Activation of escape bypass routes involving signaling pathways such as MAPK/ERK and PI3K/AKT/mTOR; 3) Dysregulation of downstream effectors; 4) Transformation into pro-metastatic phenotypes, which enable the cancer cells to survive the effects of treatment; 5) Immune adaption within the tumor microenvironment to enable cancer cell survival, via either immunedependent or immune-independent processes. 6 Theoretically, combined therapies can produce synergistic inhibition in a relatively safe manner to reduce multiple growth signal transmission responsible for the development of drug resistance, as compared to monotherapy. There is now a growing appreciation for using combination therapeutic approaches which can be exploited through multiple modalities such as radiotherapy, chemotherapy, immunotherapy, or targeted agents.
The PI3K/AKT/mTOR and MAPK/ERK signaling pathways are highly implicated in CRC pathogenesis with key mutations like RAS, BRAF, and PIK3CA arising from both pathways. There are numerous data from Phase I/II trials support the use of Palbociclib (CDK 4/6 inhibitor), [7][8][9][10] Gedatolisib (PI3K/mTOR dual inhibitors), 11,12 and PD0325901 (selective MEK1/2 inhibitor) [13][14][15] as single agents in various types of cancers. Nonetheless, these inhibitors have limited cytoreductive effect when used as single agents because of drug resistance. As shown in breast cancer models, the combination of a CDK 4/6 inhibitor with a PI3K/mTOR inhibitor have produced synergistic treatment effects. This specific drug combination can overcome treatment-related resistance by preventing RSK activation and subsequent MAPK/ERK pathway activation. 7,[16][17][18] Currently, there are several active Phase I trials evaluating the combination of Palbociclib with Gedatolisib in patients with refractory malignancies including CRC. Similarly, another Phase Ib trial is evaluating the effectiveness of combining a different PI3K/ mTOR dual inhibitor (Samotolisib) with a CDK4/6 inhibitor (Abemaciclib) in multiple common cancers. 19 By extrapolating the available literature, we believe the approach of using Palbociclib with Gedatolisib to prevent the emergence of resistance in breast cancer is hypothetically applicable to other cancers including CRC. In parallel, there is strong preclinical evidence for the evaluation of co-inhibition using Palbociclib with PD0325901 in CRC. [13][14][15] Of note, the combination of Gedatolisib with PD0325901 has previously been shown to have unacceptable toxicity in humans. 20 In summary, treatment with a targeted therapy combination has multiple advantages over treatment with a single agent as anti-proliferative efficacy can be maximized within an acceptable overlapping drug toxicity limit. In comparison to monotherapy, combined drug therapies inhibit multipletargets and have diverse cellular regulatory actions and are thus more likely to be effective in attenuating drug resistance pathways. This method has been exploited in various cancers, particularly in breast cancer. As we have noted, there is a research-gap in CRC, therefore there is a need to explore novel therapeutics using combinative drug approaches. We hypothesized that the combination of Palbociclib with either Gedatolisib or PD0325901 could produce synergistic benefits and have potential clinical relevance for the treatment of refractory CRC.

Effect of Palbociclib in combination with Gedatolisib or PD0325901 in CRC cells lines
Drug combination analysis showed that the combination of Palbociclib with Gedatolisib has a synergistic anti-proliferative effect in all CRC cell lines tested (Table 2; Figure 1). The combination of Palbociclib and Gedatolisib is highly synergistic in LS1034 cells (KRAS mutation; CI = 0.11). The combination of Palbociclib with Gedatolisib is also synergistic in DLD −1 (KRAS and PIK3CA mutated; CI = 0.58) and Caco−2 (wildtype; CI = 0.33) cells. The combination of Palbociclib with Gedatolisib is minimally synergistic in the LS411N (BRAF V600E mutated; CI = 0.64) and SNUC4 (PIK3CA mutated; CI = 0.69) cell lines.

Effect of the combination of Palbociclib with Gedatolisib on inhibition of the PI3K/AKT/mTOR pathway
We conducted RPPA analysis using 40 primary antibodies representing multiple nodes of the PI3K/AKT/mTOR, MAPK/ERK and intracellular apoptotic signaling pathways, following 4-h treatment with Palbociclib, Gedatolisib, and their combination in the Caco−2, DLD−1, LS1034, and SNUC4 cell lines. The combination Palbociclib with Gedatolisib showed synergistic inhibition of some components of the PI3K/AKT/ mTOR pathway compared to other treatment arms ( Figure 3). We did not observe increases in phosphorylated PDK1 (S241) or AKT (S473 and T308) with the combination Palbociclib with Gedatolisib, suggesting that there was no feedback activation of Table 2. Combination indexes at effective dose 50 (CI) for two drug combinations, i.e., Palbociclib with Gedatolisib (P+G) and Palbociclib with PD0325901 (P+PD) tested in this study. The CI effects are in vitro drug response in five colorectal cancer cell lines. A CI < 0.9 is indicative of a synergistic effect, between 0.9 and 1.0 is additive, and > 1.1 is antagonistic.  AKT signaling ( Figure 3). In contrast, AKT (S473) and PDK1 (S241) levels did increase in the Caco−2 and DLD−1 cells following treatment with single-agent Gedatolisib, possibly reflective of feedback loop activity. In SNUC4 cells, PDK1

Effect of the combination of Palbociclib with Gedatolisib on apoptosis and cell cycle
The expression and phosphorylation of the intracellular apoptotic signaling proteins were assessed to determine if the addition of Gedatolisib can enhance the actions of Palbociclib during cell cycle progression to increase apoptosis ( Figure 4).   50 values. The x-axis represents the combined drugs doses in the ratio of palbociclib's dose. Cell viability was assessed using a 6-day acid phosphatase assay. The graphs showed the mean cell growth ± standard error of mean (SEM) values from minimum 3 repeats in each cell lines. CI=Combination Index at effective dose 50. caspase 8, cleaved caspase 7, cleaved caspase 9, and cleaved PARP levels, possibly indicating that the observed increases were not sufficient to induce total apoptosis. However, it is possible that the 4-h treatment timepoint used in our study was too early to measure the late proteomic alterations associated with apoptosis. Of interest, the addition of Gedatolisib to Palbociclib did not induce any changes in the phosphorylated Ribosomal protein (Rb (S807/811)).

Effects of the combination of Palbociclib with Gedatolisib (P+G) on EGFR and the MAPK/ERK pathway
One of the mechanisms for the development of resistance to PI3K-targeted inhibitors is the reactivation of membrane receptor tyrosine kinases (RTKs) and/or the MAPK/ERK signaling cascade. Our RPPA data showed an increase in the total EGFR in all cell lines after 4 h of treatment ( Figure 5). Following treatment with the combination Palbociclib with Gedatolisib, EGFR levels were upregulated in all cell lines compared to vehicle control as follows: Caco−2 fold change = 2.67 ± 0.12; p < .001, DLD−1 fold change = 2.64 ± 0.49; p < .001, SNUC4 fold change = 2.39 ± 0.10; p = .001 and LS1034 fold change = 1.71 ± 0.14; p = .072. Although MAPK(T202/ Y204) phosphorylation did not increase following any treatment, MEK1/2(S217/221) phosphorylation did increase after single agent and combination treatments in DLD−1 cells, as did levels of E2F1, suggesting global activation of MAPK/ERK signaling, including with the combination treatment.   Supplementary  Table S4.

Discussion
Resistance to anti-cancer therapies evolves dynamically within 6-12 months of starting treatment due to various mechanisms, and combined drug treatment is a promising strategy for tackling resistance. 5,[7][8][9]11,21,22 In this study, we investigated the impact of combining Palbociclib (CDK 4/6 inhibitor), with either Gedatolisib (PI3K/mTOR dual inhibitor) or PD0325901 (selective MEK 1/2 inhibitor) on CRC cell line growth. We also investigated the proteomic effects of the combination Palbociclib with Gedatolisib in CRC cell lines with various mutational backgrounds to identify potential biomarker(s) for this novel therapy.
The IC 50 values for single-agent Gedatolisib were between 76 and 7200 nM. These values were comparatively higher than previous studies, which reported IC 50 values of less than 100 nM, 23,24 although Caco−2, LS411N, LS1034, and SNUC4 were not included in this analysis. We also observed a higher range of IC 50 values for single-agent Palbociclib in our cell lines, in comparison to other studies. 25 PD0325901 demonstrated a relatively low IC 50 range in our cell lines, suggesting more innate sensitivity to MEK inhibition.
Our results demonstrated that the combination Palbociclib with either Gedatolisib or PD0325901 exhibits synergistic antiproliferative effects, relative to the single agents, in all cell lines including LS1034 (KRAS mutated) and DLD−1 (co-occurring KRAS with PIK3CA mutated) cells. These are important findings since KRAS and PIK3CA mutations occur in approximately 60% and 20% of CRC, respectively. 26 In LS411N (BRAF V600E mutated) cells, the combination Palbociclib with Gedatolisib demonstrated mild synergy whilst the combination Palbociclib with PD0325901 failed to show any synergistic effects (CI = 0.64 versus 14.7). In LS1034 cells, we noted a synergistic effect with both drug combinations, however the combination Palbociclib with Gedatolisib appears to be superior (CI = 0.11 versus 0.29). In the Caco−2 wild-type cells, both drug combinations demonstrated comparable synergistic effects. Taken together, we considered the combination of Palbociclib with Gedatolisib to be of higher priority for future clinical development, compared to the combination of Palbociclib with PD0325901 and thus decided to focus our proteomic investigations on Palbociclib with Gedatolisib. The subnanomolar IC 50 range of Gedatolisib makes it a more favorable companion drug than PD0325901 to avoid excessive overlapping toxicities. Furthermore, strong efficacy and safety data is already available from Phase I clinical trials involving the combination Palbociclib with Gedatolisib. 7,8 In these trials, Palbociclib (125 mg) was administered orally, daily for 3 weeks with Gedatolisib (110 mg) administered intravenously once during the 4-week cycle.
In our RPPA study following 4 h of exposure to the combination Palbociclib with Gedatolisib, we observed suppression of S6rp (S240/S244) across all cell lines. There was also a significant suppression of S6rp (S235/S236) in Caco−2, DLD−1, and LS1034 cells. This suppression was much more marked with the combination Palbociclib with Gedatolisib than with either single agent alone. Suppression of S6rp was not associated with any increased expression or phosphorylation of AKT, indicating that there was no upstream PI3K reactivation in response to the combination. We thus believe that the combination Palbociclib with Gedatolisib acts primarily at the level of mTOR, which is a downstream effector of the PI3K/AKT signaling cascade. Our results also show that the combination Palbociclib with Gedatolisib likely exhibits stronger inhibition of the PI3K/AKT/mTOR pathway in comparison to control or single-agent therapy. For example, we observed increased levels of AKT (S473) and PDK1 (S241) in some cell lines with single-agent treatment, possibly reflective of feedback loop activity.
In view of the global suppression of S6rp (S240/S244) in all tested cell lines, this may be a promising predictive marker of clinical responsiveness for this combination therapy. As reported by Iwenofu et al.,27 S6rp is considered a better surrogate biomarker of mTOR activity in comparison to p70S6K, also known as Ribosomal protein S6 kinase beta−1 (S6K1). This is because p70S6K has structural similarity to p90S6K, which is not phosphorylated by mTOR. 27,28 As a key component of the PI3K/AKT signaling cascade, mTOR plays a crucial role in the regulation of energy metabolism and protein synthesis by directly activating p70S6K. 29-32 p70S6K is a serine-threonine kinase that controls S6rp phosphorylation at five serine residues (S235, S236, S240, S244, and S247), leading to initiation of protein synthesis. 33 In contrast to the S240/244 residues which are solely regulated by p70S6K, phosphorylation at the S235/S236 residues is controlled by multiple kinases including p70S6K, p90RSK, and PKA. 34 This may explain the suppression of S6rp (S235/S236) which was significant in Caco−2, DLD−1, and LS1034 but not in SNUC4 cells, in contrast to S6rp (S240/S244).
Emerging experimental data has suggested utilizing PI3K/ mTOR inhibitors to induce non-cell autonomous actions by modulating signal transduction during G 1 to S phases, leading to increased cell death. [35][36][37][38] Interestingly, we did not observe any increase in pRb (S807/S811) following treatment with the combination Palbociclib with Gedatolisib. This is consistent with results previously described by Vora et al. 39 It appears that PI3K inhibition suppress AKT phosphorylation but can fail to suppress CDK 4/6 activity, as measured by Rb phosphorylation. 40 Nonetheless, the regulation of Rb function by phosphorylation during cell cycle is not fully understood. Rb in mammalian cells has 15 known phosphorylation sites and it appears that Rb phosphorylation at specific sites is required for Rb to regulate apoptosis. 41,42 In vitro studies have shown that phosphorylation of Rb at S608/S795 in addition to S807/S811 may play a role in the induction of apoptosis. 43 Furthermore, there is evidence to suggest that dephosphorylation of Rb has been widely observed during apoptosis. 44 This may explain the equivocal level of pRb (S807/S811) we observed in our RPPA analysis.
Unlike the single agents, the combination Palbociclib with Gedatolisib induced (early) pro-apoptotic effects, as demonstrated by increased BAX and Bcl−2 levels in most cell lines tested. The increase in both markers was significant in DLD−1 and SNUC4 cells but was not significant in LS1034 cells, suggesting that the magnitude of effect may be dependent on the cells' mutational status. We did not observe any increase in caspase 3, caspase 8, cleaved caspase 7, cleaved caspase 9, or cleaved PARP levels, which are indicative of total apoptosis. However, it is possible that the 4-h timepoint used for our analysis was too early to evaluate the proteomic alterations related to the late stage of apoptosis.
Finally, we observed EGFR and RSK upregulation in all cell lines after 4 h of drug treatment, which may be associated with upstream MAPK/ERK reactivation. Total EGFR and RSK upregulation were observed with both mono-and combination therapy in some of the cell lines. This suggests that the mechanism promoting resistance to PI3K-targeted inhibitors (which was Gedatolisib in this study) include feedback loops, which lead to reactivation of membrane RTKs and the contralateral MAPK/ERK pathway. This further supports our hypothesis that a multiple target inhibition strategy, rather than single agent, is a better therapeutic option to prevent the development of resistance. The combined drug inhibition using Palbociclib with Getatolisib would simultaneously target the upstream and downstream effectors including the interconnection points between the PI3K/AKT/mTOR and MAPK/ERK pathways. It provides broader inhibition on the vast interconnection of both pathways, while minimizing feedback loops activation.
It is important to note the limitations of our study. First, it was an in vitro study and limited to five cell lines. Second, not all specific exon mutations were tested in this study, specifically PIK3CA mutations in exon 20 which may be biologically more relevant than exon 9 mutations from an epidemiology standpoint. [45][46][47] Third, the RPPA analysis with 40 preselected antibodies was performed at only two timepoints (i.e., . 30 minutes and 4 h) post-drug exposure. The RPPA results are dependent on the selected timepoints, and it is likely we will be able to capture additional proteomic information if longer timepoints are used. The specific mechanism of synergism for the combination Palbociclib with Gedatolisib could not be completely defined in this study; however, from our RPPA analysis there are several possible mechanisms, including more complete inhibition of protein synthesis-related signaling (e.g., S6rp(S240/S244)) and increased activation of early apoptotic signaling. Despite these limitations, our study has produced evidence to support further in vivo evaluation, which is in progress.
In summary, the novel combination Palbociclib with Gedatolisib displays clear synergistic anti-proliferative effects in both wild-type and mutated CRC cell lines, relative to the single agents. Our results offer good rationale for further in vivo study and clinical development of Palbociclib and Gedatolisib as emerging therapeutics in metastatic CRC patients. S6rp (S240/S244) may be a marker of responsiveness for this novel combination therapy.

Cell lines
Five human CRC epithelial cell lines with commonly found mutational variations were used in this study (Table 1). Caco  Supplementary  Table S1.

Proliferation assays and drug combination analysis
The acid phosphatase assay was used to test the antiproliferative effects of Palbociclib, Gedatolisib and PD0325901, alone and in combination in each cell line. This was performed over 6 days period, as previously described. 48 Cells were plated at 1 × 10 4 cells/mL into 96well plates (100 µL per well) and incubated at 37°C with 5% CO 2 for 24 h. Two hundred microliters of sterile H 2 O was added around the edges of the plate to prevent it from drying out. Following 24-h incubation, drugs were added at the indicated concentrations and incubated at 37°C for 5 days (120 h). On Day 6, all the drug was removed, washed, and processed for absorbance measurement at 405 nm using a 96-well plate reader. Inhibition of proliferation was calculated relatively to untreated controls to obtain the dose of half maximal inhibitory concentration (IC50). A minimum number of triplicate biological assays was performed for each experiment. IC50 values were calculated using CacuSyn software. The individual inhibitor IC50 values were used for dosing guidance in the subsequent drug combination analysis. The same cell number (1×10 4 cells/mL) was used for the drug combination analysis. Drug concentrations used for the combination assays are shown in Table 1.

Protein extraction and Reverse Phase Protein Array (RPPA)
4 × 10 5 cells were seeded in 6-well plates and grown until confluent. The total protein was extracted using 100 µl lysis buffer (15% NACL 1 M, 1% Triton-X 100, 5% TRIS, 14% phosphatase inhibitors 7×, 65% dH 2 O), as previously described by us. 49,50 Protein was quantified by the bicinchoninic acid (BCA) assay and stored at −80•C until analysis. The combination Palbociclib with Gedatolisib was selected for RPPA investigation because this combination demonstrated synergism in all cell lines tested in our study.
Cell lysates with a protein concentration of 1.5 µg/µL for each replicate were prepared. Before RPPA processing, each sample was solubilized in sodium dodecyl sulfate (SDS) sample buffer (40% Glycerol, 8% SDS, 0.25 M Tris-HCL, pH 6.8, 50 nM Bond-breaker TCEP solution) and heated to 80°C for 5 min. RPPA analysis was carried out using triplicate biological replicates following 4-h treatment with Palbociclib, Gedatolisib, and their combination in Caco−2, DLD−1, LS1034, and SNUC4 cell lines. The cell lines were treated at the same fixed ratio doses used in the drug combination proliferative analysis. Details of all primary antibodies used for RPPA analysis are detailed in Supplementary Table S2.

Statistical analysis
Microsoft Excel software was used to record all the raw datasets. CalcuSyn software version 3.1 (Biosoft) was used to calculate IC 50 and combination index at effective dose 50 (CI) values. The CI values were determined using the Chou-Talalay equation on CalcuSyn. A CI < 0.9 is considered synergistic, between 0.9 and 1.0 is additive and > 1.1 is antagonistic. Each experiment was repeated 3-4 times. For the RPPA experiments, the mean and standard error of mean (SEM) were calculated from three biologically independent protein samples analyzed on the same RPPA slide. The mean and SEM were normalized to the vehicle-treated control samples. Based on our internal precision studies, this allows us to detect changes in protein expression with a coefficient of variance (CV) of less than 20%. 51 To evaluate the effect of the combination treatment, a one-way analysis of variance (ANOVA) with Tukey's multiple-comparison test was used (GraphPad PRISM version 8). To compare the effects of Palbociclib, Gedatolisib, and their combination treatment on protein expression and phosphorylation, the Kruskal-Wallis non-parametric test was used. P values of < 0.05 were considered statistically significant.

Author contributions
BH and ST conceived the study and co-ordinated the experiments and revised the manuscript. CLL coordinated the experiments, performed the experiments, analyzed the data, and wrote the manuscript. RA performed parts of the drug combination cell assays. MC carried out the RPPA experiments. SM performed statistical analyses. SK, AC, AF, and JW performed the cell protein lysate experiments. BON performed data analysis. All authors read and approved the final manuscript.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This work was supported by the North East Cancer Research and Education Trust (NECRET) and St. Luke's Institute for Cancer Research (SLICR)

Notes on contributors
Dr. Cha Len Lee, is a translational oncologist with a particular interest in gastrointestinal cancers. She is currently a Medical Oncology fellow in Princess Margaret Cancer Centre, Toronto, Canada.
Dr. Mattia Cremona, is a molecular biologist, with a special interest in translational oncology research. Dr. Cremona's research involves using proteomic high-throughput approaches to study the status of the proteins inside cancer cells and to highlight changes with normal cells that could explain why patients do not respond to therapy or develop resistance after an initial response.
Dr. Angela Farrelly, graduated from the University of Nevada with a PhD in Molecular Physiology and Pharmacology in 2004. Her research interests focus on investigating the sensitivity of cancer cell lines to chemotherapy agents, novel targeted therapies and combinations of these agents.
Ms. Julie Workman, is a molecular scientist with a particular interest in translational oncology. Her research interests include preclinical investigation of novel targeted therapies in GI cancers.
Dr. Sean Kennedy, is a translational scientist with a particular interest are in protein-protein interaction technologies and deciphering the complex systems, influencing cancer therapeutic resistance.
Dr. Razia Aslam, is a translational oncologist with a particular interest in novel combination therapies to treat cancers, in particular colorectal cancer.
Dr. Aoife Carr, is a biomedical scientist who has a particular interest in targeting the PI3K pathway in colorectal cancer.

Dr.
Stephen Madden, is a biostatistician whose research interests are in cancer genomics and transcriptomics, next generation sequence analysis, multivariate statistics and data integration.

Dr. Brian O'
Neill, is a radiation oncologist with a expertise in Prostate and Bladder cancers; Oesophageal, Stomach, Rectal and Anal cancers and Lung Cancer.
Professor Bryan Hennessy, is a clinician scientist whose research team has had a considerable impact on the fields of kinase signalling. He is also an international leader in the application of reverse phase protein arrays (RPPA) for quantitative protein profiling to interrogate predictive and prognostic markers in breast, colon and other cancers.
Dr. Sinead Toomey, is a translational oncology research scientist with a particular interest in identifying novel therapeutic targets in cancers and preclinical research using combinations of targeted therapies to overcome resistance.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material. Further data are available from the corresponding author upon request.